We have constructed an operational epitope map of the type 3 parainfluenza virus (PIV3) hemagglutinin-neuraminidase (HN) protein. Neutralizing monoclonal antibodies (mAbs) specific for the HN protein were used to select antibody-resistant antigenic variants. Reactivity patterns of the mAbs with the antigenic variants and with human clinical strains have identified 11 unique HN epitopes. Five of these epitopes vary antigenically in clinical strains whereas variation in six other epitopes is extremely rare. Competitive-binding radioimmunoassays indicated that the 11 epitopes are located in two topologically distinct antigenic sites on the HN molecule. The antigenic variants achieve maximum levels of replication in cotton rat lungs and nasal turbinates which are not significantly different from that achieved by wild type PIV3, indicating that the HN molecule can accommodate antigenic alterations without loss of function. Passive transfer of individual mAbs that neutralize virus in vitro causes a 10-100 fold reduction in viral replication in cotton rat lungs after challenge with wild-type virus, whereas transfer of a mixture of six mAbs reduces virus replication to an undetectable level. Nucleotide sequence analysis of the antigenic variants is being performed to construct a molecular epitope map of the HN protein. Comparison of the HN gene sequences of three variants to that of the wild type virus identified a single point mutation in each of the variant HN genes. These mutations code for a single amino acid substitution in the HN protein which is responsible for loss of antibody-binding.